Abstract: The oral part of the pontine reticular formation (PnO) is a component of the ascending reticular activating system and plays a role in the regulation of sleep and wakefulness. The PnO receives glutamatergic and GABAergic projections from many brain regions that regulate behavioral state. Indirect, pharmacological evidence has suggested that glutamatergic and GABAergic signaling within the PnO alters traits that characterize wakefulness and sleep. No previous studies have simultaneously measured endogenous glutamate and GABA from rat PnO in relation to sleep and wakefulness. The present study utilized in vivo microdialysis coupled on‐line to capillary electrophoresis with laser‐induced fluorescence to test the hypothesis that concentrations of glutamate and GABA in the PnO vary across the sleep/wake cycle. Concentrations of glutamate and GABA were significantly higher during wakefulness than during non‐rapid eye movement sleep and rapid eye movement sleep. Regression analysis revealed that decreases in glutamate and GABA accounted for a significant portion of the variance in the duration of non‐rapid eye movement sleep and rapid eye movement sleep episodes. These data provide novel support for the hypothesis that endogenous glutamate and GABA in the PnO contribute to the regulation of sleep duration.